CN104892729B - Sphyrna lewini cartilage angiogenesis inhibiting factor - Google Patents

Abstract

The invention relates to a Sphyrna lewini angiogenesis inhibitor, a preparation method and application thereof, wherein the amino acid sequence of the angiogenesis inhibitor is Pro-Asp-Tyr-Lys-Phe-Lys, and the ESI-MS detected molecular weight is 796.90 Da. The angiogenesis inhibiting factor can effectively inhibit the angiogenesis of chick chorioallantoic membrane, and has obvious inhibiting effect on the expression of three angiogenesis promoting factors, namely vascular endothelial cell growth factor, basic fibroblast growth factor and platelet-derived growth factor of lung cancer tissues of Lewis lung cancer-bearing mice.

Description

An inhibitor of cartilage angiogenesis in hammerhead shark

An inhibitor of cartilage angiogenesis in hammerhead shark

technical field

The invention relates to an vascular inhibitory factor extracted from aquatic organisms, in particular to an angiogenesis inhibitory factor of hammerhead shark cartilage.

Background technique

Angiogenesis is one of the important pathological features of angioproliferative diseases such as tumors, diabetic retinopathy, rheumatoid arthritis and chronic inflammation. The anti-angiogenesis-based tumor biotherapy research has become a research hotspot of anti-tumor drugs in the past decade.

Compared with chemotherapy aimed at directly killing tumor cells, tumor angiogenesis inhibitor (TAI) has the following unique advantages: (1) TAI directly acts on vascular endothelial cells, while anti-cancer drugs are affected by tissue diffusion. Due to the influence of tissue fibrosis and necrosis, the effective drug concentration in the tissue is often not reached; (2) Compared with genotype unstable tumor cells, vascular endothelial cells are normal cells with stable genotype and are not easy to develop drug resistance; ( 3) The vascular endothelial cells of the primary tumor and the secondary tumor are the same, but the biological characteristics of the tumor cells in the primary tumor and the secondary tumor are quite different, and the response to chemotherapy is also different; (4) The proliferation rate of tumor vascular endothelial cells is higher than that of the secondary tumor. Normal tissue is many times faster and TAI has little effect on normal tissue. Based on the above reasons, anti-angiogenic tumor therapy strategies will play an important role in tumor therapy in the future.

The applicant's research found that the research on the preparation of angiogenesis inhibitory factors using hammerhead shark cartilage as a raw material is at a blank stage, and the application of hammerhead shark cartilage angiogenesis inhibitory factors in the preparation of drugs for inhibiting angiogenesis and preventing and treating tumors There are no reports.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a cartilage angiogenesis inhibitor of hammerhead shark, which exhibits a significant angiogenesis inhibitory activity.

The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: an angiogenesis inhibitor of hammerhead shark cartilage, and the amino acid sequence of the angiogenesis inhibitor is Pro-Asp-Tyr-Lys-Phe-Lys (PDYKFK) , and the molecular weight detected by ESI-MS was 796.90 Da.

The preparation method of the cartilage angiogenesis inhibitory factor of hammerhead shark can be prepared by the following steps:

1) Preparation of active protein components of hammerhead shark cartilage: The crushed and homogenized hammerhead shark cartilage was added to 1.0 mol/L guanidine hydrochloride solution at a solid-liquid ratio of 1 g: 8 to 10 mL, and the solution was shaken at 4°C. After 32-36 hours of extraction, centrifuge at 4 °C and 10 000 r/min for 15-20 min, take the supernatant and put it into a dialysis bag with a molecular weight cut-off of 1 kDa, and dialyze it with Tris-HCl (pH 7.6) buffer at below 4 °C 22-24h, get the crude extract of hammerhead shark cartilage protein; place the crude extract of hammerhead shark cartilage protein at 4°C, slowly add pre-cooled acetone below 4°C until the acetone concentration is 30%, and let stand for 0.5 After ~1h, centrifuge at 10000r/min below 4℃ for 15~25min, take the supernatant and add pre-cooled acetone below 4℃ until the acetone concentration reaches 60%. Centrifuge for 15-25min, take the precipitate and place it in a dialysis bag with a molecular weight cut-off of 1kDa and dialyze it with double distilled water below 4°C for 22-24h, and freeze-dry the dialysate to obtain the active protein component of hammerhead shark cartilage;

2) Enzymatic hydrolysis of the active protein component of hammerhead shark cartilage: add the active protein component of hammerhead shark cartilage to Gly-NaOH buffer (0.05mol/L, pH 9) at a solid-liquid ratio of 1 g: 20-25 mL. ～10) to obtain a mixed solution; the temperature of the mixed solution was raised to 45～55℃ and preheated for 5～10min, and alkaline protease (enzyme activity≥2.0%) was added according to 1.5～2.5% of the mass of the active protein components of hammerhead shark cartilage. ×10 ⁵ U/g), the enzymatic hydrolysis temperature was 45-55 °C, after 5-6 h of enzymatic hydrolysis, the solution was heated to 90-95 °C, and after maintaining the temperature for 10-15 min, centrifuged at 10 000 g for 20-25 min, Take the supernatant, which is the enzymatic hydrolysis product of the cartilage active protein component;

3) Preparation of hammerhead cartilage angiogenesis inhibitory factor: the prepared cartilage active protein component enzymolysis product is subjected to ultrafiltration treatment with a 1kDa ultrafiltration membrane, and the fraction with a molecular weight less than 1kDa is collected to obtain an ultrafiltration enzymatic hydrolyzate, and then The enzymatic hydrolysate was purified by gel filtration chromatography, cell membrane chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) successively to obtain angiogenesis inhibitory factor of hammerhead shark cartilage.

Preferably, the hammerhead shark in the step 1) is the hammerhead shark Sphyrna lewini.

As an improvement, the specific process of gel filtration chromatography, cell membrane chromatography and RP-HPLC purification in the step 3) is:

Gel filtration chromatography: The above-mentioned ultrafiltration enzymolysis solution was prepared into a solution of 15-25 mg/mL with pH 6.5-7.5 phosphate buffer, and separated by Sephadex G-15 column chromatography (2.6×80cm) , eluted with pH 6.5-7.5 phosphate buffer, and collected the eluted fractions according to the absorbance curve at 215 nm. Among them, the fraction with the highest inhibitory effect on the angiogenesis of chick chorioallantoic membrane (CAM) is gel chromatography Enzymatic hydrolyzate.

Cell Membrane Chromatography Purification: The above gel chromatography enzymatic hydrolysate was made into a solution of 40-50 μg/mL with three-distilled water, added to the cell membrane chromatography column for purification, and the eluted fractions were collected according to the absorbance curve at 215 nm. Chick embryo chorioallantoic membrane (CAM) angiogenesis-inhibitory component was the highest component of cell membrane chromatography-purified enzymatic hydrolysate.

RP-HPLC purification: The above-mentioned cell membrane chromatography-purified enzymatic hydrolysate was prepared into a solution of 80-100 μg/mL with three-distilled water, and purified by RP-HPLC. According to the inhibitory effect on chicken embryo chorioallantoic membrane (CAM) angiogenesis One highly active polypeptide, Pro-Asp-Tyr-Lys-Phe-Lys (PDYKFK), has a molecular weight of 796.90 Da detected by ESI-MS.

Preferably, the cell membrane chromatography conditions are: the injection volume is 5-10 μL; the cell membrane chromatography column (150 mm×4.6 mm, 5 μm); the column temperature is 37° C.; the mobile phase: three-distilled water; the ultraviolet detection wavelength is 215 nm; /min.

Preferably, the RP-HPLC conditions are: the injection volume is 15-20 μL; the chromatographic column is Zorbax C18 (250 mm×4.6 mm, 5 μm); the mobile phase is 20% acetonitrile; and the ultraviolet detection wavelength is 215 nm. Further preferably, the cell membrane used in the cell membrane chromatography column is the cell membrane of the human umbilical vein endothelial cell line ECV304.

Compared with the prior art, the present invention has the advantages that the extraction and purification processes are more advanced, the prepared angiogenesis inhibitory factor has significant activity, and can be used for the prevention and treatment of tumor diseases.

Description of drawings

Fig. 1 is the Sephadex G-15 column chromatography chromatogram of the ultrafiltration enzymolysis solution (≤1kDa component) of the present invention;

Fig. 2 is the cell membrane chromatogram of the enzyme hydrolysate (Fr.C) prepared by Sephadex G-15 of the present invention;

Fig. 3 is the RP-HPLC chromatogram of the cell membrane chromatography-purified enzymatic hydrolysate (Fr.C-II) of the present invention.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments of the accompanying drawings.

Example: a preparation method of hammerhead shark angiogenesis inhibitory factor, the preparation process is as follows: hammerhead shark cartilage → tissue fragmentation → guanidine hydrochloride extraction → acetone fractional precipitation → gel filtration chromatography purification → cell membrane chromatography Purification→High Performance Liquid Chromatography Purification→Angiogenesis Inhibitor.

1) Preparation of active protein components of hammerhead shark cartilage: the crushed and homogenized hammerhead shark (Sphyrnalewini) cartilage was added to a 1.0 mol/L guanidine hydrochloride solution at a solid-liquid ratio of 1 g: 10 mL, at 4° C. After shaking extraction for 36h, centrifuge at 4°C and 10 000 r/min for 20min, take the supernatant and put it into a dialysis bag with a molecular weight cut-off of 1kDa, and use Tris-HCl (pH 7.6) buffer solution at below 4°C for 24h dialysis. Get the crude extract of hammerhead shark cartilage protein; place the crude extract of hammerhead shark cartilage protein at 4 °C, slowly add pre-cooled acetone below 4 °C to the acetone concentration of 30%, let stand for 1 hour, and then put it at 4 °C. Centrifuge at 10000 r/min below ℃ for 25 min, take the supernatant and add pre-cooled acetone below 4 ℃ until the acetone concentration reaches 60%. After standing for 1 h, centrifuge below 4 ℃ and 10 000 r/min for 25 min. The 1kDa dialysis bag was dialyzed with double distilled water below 4°C for 24h, and the dialysate was freeze-dried to obtain the active protein component of hammerhead shark cartilage;

2) Enzymatic hydrolysis of the active protein component of hammerhead shark cartilage: add the active protein component of hammerhead shark cartilage to Gly-NaOH buffer (0.05mol/L, pH 9-10) at a solid-to-liquid ratio of 1g:25mL. ) to obtain a mixed solution; the temperature of the mixed solution was raised to 45-55 °C and preheated for 5 min, and alkaline protease (enzyme activity ≥ 2.0×10 ⁵ U/g) was added according to 1.8% of the mass of the active protein component of hammerhead shark cartilage. ), the enzymatic hydrolysis temperature was 50 °C, after 5 h of enzymatic hydrolysis, the solution was heated to 95 °C, and after maintaining this temperature for 12 min, centrifuged at 10 000 g for 25 min, and the supernatant was taken, which is the enzymatic hydrolysis product of the cartilage active protein component;

3) Preparation of hammerhead cartilage angiogenesis inhibitory factor: the prepared cartilage active protein component enzymolysis product is subjected to ultrafiltration treatment with a 1kDa ultrafiltration membrane, and the fraction with a molecular weight less than 1kDa is collected to obtain an ultrafiltration enzymatic hydrolyzate, and then The enzymatic hydrolysate was purified by gel filtration chromatography, cell membrane chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) successively to obtain angiogenesis inhibitory factor of hammerhead shark cartilage.

①Gel filtration chromatography: The above-mentioned ultrafiltration enzymatic hydrolyzate was prepared into a solution of 25 mg/mL with pH 7.0 phosphate buffer, separated by Sephadex G-15 column chromatography (2.6×80cm), and the solution was separated with pH 7.0. 7.0 phosphate buffer was used for elution, and the elution fractions were collected according to the absorbance curve at 215 nm. Among them, the strongest inhibitory effect on the angiogenesis of the chick embryo chorioallantoic membrane (CAM) was the gel chromatography enzymatic hydrolysate Fr. .C (Figure 1).

② Purification by cell membrane chromatography: The above gel chromatography enzymatic hydrolysate Fr.C was prepared into a solution of 50 μg/mL with three distilled water, and added to the cell membrane chromatography column for purification (conditions: injection volume 50 μL; human umbilical vein endothelial cells The cell membrane column of strain ECV304 (150mm×4.6mm, 5μm); the column temperature is 37°C; mobile phase: three-distilled water; UV detection wavelength: 215nm; flow rate: 0.2mL/min), among which, for chicken embryo chorioallantoic membrane ( CAM) with the strongest inhibitory effect on angiogenesis was the cell membrane chromatography-purified enzymatic hydrolyzate Fr.C-II (Fig. 2).

③ RP-HPLC purification: The above-mentioned cell membrane chromatography-purified enzymatic hydrolysate Fr.C-II was made into a solution of 80-100 μg/mL with three distilled water, and purified by RP-HPLC (condition: the injection volume was 15 μL; the chromatographic column was Zorbax C18 (250mm×4.6mm, 5μm); mobile phase is 20% acetonitrile; UV detection wavelength is 215nm), according to the inhibition of chicken embryo chorioallantoic membrane (CAM) angiogenesis, a highly active polypeptide was obtained.

④Structure detection: collect the polypeptide with the strongest inhibitory effect on the angiogenesis of chicken embryo chorioallantoic membrane (CAM), which is detected as a single peak, and the amino acid sequence determined by protein/peptide sequence analyzer is Pro-Asp-Tyr-Lys-Phe -Lys(PDYKFK), the molecular weight detected by ESI-MS is 796.90Da.

The obtained Pro-Asp-Tyr-Lys-Phe-Lys (PDYKFK) was tested for the angiogenesis inhibitory activity of chicken embryo chorioallantoic membrane (CAM). Allantoic Membrane Technology - Airless Chamber Incubation" (recorded in "Journal of Sun Yat-Sen University (Natural Science Edition)", 2003, Volume 2 (Volume 42): pp. 126-128). The results showed that the angiogenesis inhibitor PDYKFK of hammerhead shark significantly inhibited the angiogenesis of chick chorioallantoic membrane (CAM) in a dose-dependent manner (Table 1).

Table 1 Inhibitory effect of hammerhead shark angiogenesis inhibitor on angiogenesis in chick embryo chorioallantoic membrane (CAM)

The obtained PDYKFK was subjected to the determination of the influence of the expression of pro-angiogenic factors, and the determination method was referred to "Experimental Study on Scorpion Venom Polypeptide Extract Combined with Chemotherapy Inhibiting Lewis Lung Cancer Angiogenesis" (recorded in "Chinese Journal of Traditional Chinese Medicine", 2011, No. 12 (Vol. 36: pp. 1644-1649). The angiogenesis inhibitor PDYKFK (20.0 mg/kg·d×14) was intraperitoneally injected into Lewis lung cancer-bearing mice, and the lung cancer tissues were taken for immunohistochemical examination. The factor PDYKFK significantly inhibited the expression of three pro-angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) (Table 2).

Table 2 Inhibitory effect of hammerhead shark angiogenesis inhibitor on the expression of pro-angiogenic factor

Although the present invention has been described in conjunction with preferred embodiments, it is not intended to limit the present invention, and any person skilled in the art, without departing from the spirit and scope of the present invention, can implement various aspects of the subject matter set forth herein. Therefore, the protection scope of the present invention should be determined by the scope defined by the appended claims.

SEQUENCE LISTING

<110> Zhejiang Ocean University

<120> An inhibitor of cartilage angiogenesis in hammerhead shark

<130> zjou-2015-wb0507

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 6

<212> PRT

<213> Synthetic

<400> 1

Pro Asp Tyr Lys Phe Lys

1 5

Claims (1)

1. The Sphyrna lewini angiogenesis inhibitor is characterized in that the amino acid sequence of the Sphyrna lewini angiogenesis inhibitor is Pro-Asp-Tyr-Lys-Phe-Lys, and the molecular weight of ESI-MS detection is 796.90 Da.

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